Signal transduction mechanisms to myosin phosphatase

肌球蛋白磷酸酶的信号转导机制

• 批准号: 8436884
• 负责人: JAMES T STULL
• 金额: $ 39.75万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-01-15 至 2016-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8436884
• 关键词: Ablation; Adult; Affect; Alleles; Area; Asthma; Binding; Biochemical; Biological Models; Calcium; Calmodulin; Carbachol; Cell Surface Receptors; Cell division; Cell-Matrix Junction; Cells; Chemicals; Clinical; Contractile Proteins; Development; Disease; Event; Genes; Genetic; Hormones; Hypertension; Inflammatory Bowel Diseases; Inflammatory disease of the intestine; Intestines; Investigation; Knock-out; Knowledge; Light; Measurement; Measures; Mechanics; Microfilaments; Molecular; Molecular Motors; Motor; Movement; Mus; Muscarinic M3 Receptor; Muscle Contraction; Mutate; Myocardium; Myosin ATPase; Myosin Light Chain Kinase; Myosin Light Chains; Myosin Regulatory Light Chains; Myosin Type II; Nerve; Neurotransmitters; Pathology; Pathway interactions; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphorylation Site; Phosphotransferases; Play; Process; Protein Dephosphorylation; Protein Kinase; Protein Kinase C; Proteins; Regulation; Relative (related person); Role; Signal Pathway; Signal Transduction; Signal Transduction Pathway; Signaling Protein; Skeletal Muscle; Skeletal Muscle Myosins; Smooth Muscle; Smooth Muscle Myocytes; Smooth Muscle Myosins; Tamoxifen; Testing; Tissues; Trachea; Transgenic Organisms; Translational Research; base; cell motility; cytokine; dimer; electric field; ileum; in vivo; indexing; inhibitor/antagonist; kinase inhibitor; mature animal; myosin phosphatase; non-muscle myosin; novel; public health relevance; respiratory smooth muscle; response; scaffold; success

DESCRIPTION (provided by applicant): Activation of many cell surface receptors initiates diverse cellular movements such as cell migration, cell- matrix adhesion and contraction. These movements respond to increased cytosolic Ca2+ concentrations ([Ca2+]i) and activation of Ca2+/calmodulin (CaM)-dependent myosin light chain kinase (MLCK). MLCK phosphorylates myosin regulatory light chain subunit (RLC), allowing myosin to bind actin filaments for contraction. Signaling pathways are proposed for inhibition of MLCP activity which increases RLC phosphorylation (Ca2+-sensitization). MLCP subunit MYPT1 and the inhibitor protein CPI-17 may be phosphorylated by different Ca2+-independent kinases. Based on our recent successes in using molecular transgenic and conditional gene ablation approaches to establish MLCK's role in Ca2+-dependent signaling in mice, we propose similar approaches to unravel integrative signaling pathways in relation to MLCP and Ca2+- sensitization mechanisms. Specific Aim 1: Is the regulatory subunit of myosin light chain phosphatase (MYPT1) necessary for effective signaling to RLC phosphorylation? We will knock out MYPT1 in adult mice containing floxed MYPT1 alleles by tamoxifen-controlled Cre expression specifically in smooth muscle cells. Gross pathology will be assessed in phasic (ileum) and tonic (trachea) smooth muscles, including histological analyses and expression of contractile and signaling proteins. Carbachol and electric field stimulation (EFS) eliciting responses from parasympathetic nerves will induce temporal cellular changes in [Ca2+]i, RLC phosphorylation and contraction. We will assess Ca2+-sensitization indices by measuring MYPT1 Thr696 and Thr853 phosphorylation in control tissues in addition to CPI-17 phosphorylation. We will test the hypothesis that MYPT1 is necessary for robust RLC dephosphorylation. We will also determine the relative importance of CPI-17 phosphorylation compared to MYPT1 phosphorylation in the Ca2+-sensitization response induced by muscarinic M3 receptors. Specific Aim 2: How does MYPT1 regulate MLCP activity to sustain RLC phosphorylation during Ca2+-sensitization? We will mutate the two regulatory phosphorylation sites in MYPT1 (Thr696Ala and Thr853Ala) individually or together, and measure biochemical and cellular responses as described in Aim 1. We will thus test the hypothesis that both MYPT1 Thr696 and Thr696 phosphorylation are important for Ca2+-sensitization responses. Specific Aim 3: Does Ca2+-independent ZIPK affect Ca2+- sensitizing MYPT1 and CPI-17 phosphorylation? Mice containing floxed genes for ZIPK will be used for gene ablation in adult animals because there are no selective pharmacological inhibitors for this kinase. We will test the hypothesis that the Ca2+-independent kinase ZIPK phosphorylates MYPT1 and CPI-17 as an integral component of the Ca2+-sensitization process.

描述（由申请人提供）：许多细胞表面受体的激活启动不同的细胞运动，如细胞迁移、细胞-基质粘附和收缩。这些运动响应于胞质Ca 2+浓度（[Ca 2 +]i）的增加和Ca 2 +/钙调蛋白（CaM）依赖性肌球蛋白轻链激酶（MLCK）的激活。MLCK磷酸化肌球蛋白调节轻链亚基（RLC），使肌球蛋白结合肌动蛋白丝进行收缩。提出了抑制MLCP活性的信号传导途径，其增加RLC磷酸化（Ca 2 +-敏化）。MLCP亚基MYPT 1和抑制蛋白CPI-17可能被不同的Ca 2+非依赖性激酶磷酸化。基于我们最近成功地使用分子转基因和条件性基因消融方法建立MLCK在小鼠Ca 2+依赖性信号传导中的作用，我们提出了类似的方法来解开与MLCP和Ca 2+致敏机制相关的整合信号传导途径。具体目标1：肌球蛋白轻链磷酸酶（MYPT 1）的调节亚基是否是RLC磷酸化的有效信号所必需的？我们将敲除MYPT 1在成年小鼠含有floxed MYPT 1等位基因的他莫昔芬控制的Cre表达，特别是在平滑肌细胞。将评估阶段性（回肠）和强直性（气管）平滑肌的大体病理学，包括组织学分析以及收缩和信号蛋白的表达。卡巴胆碱和电场刺激（EFS）引起副交感神经的反应，将诱导细胞[Ca 2 +]i、RLC磷酸化和收缩的暂时变化。我们将通过测量对照组织中MYPT 1 Thr 696和Thr 853磷酸化以及CPI-17磷酸化来评估Ca 2+致敏指数。我们将检验MYPT 1对于稳健的RLC去磷酸化是必需的这一假设。我们还将确定CPI-17磷酸化与MYPT 1磷酸化在毒蕈碱M3受体诱导的Ca 2+致敏反应中的相对重要性。具体目标2：MYPT 1如何调节MLCP活性以维持钙致敏过程中的RLC磷酸化？我们将单独或一起突变MYPT 1中的两个调节磷酸化位点（Thr 696 Ala和Thr 853 Ala），并测量目标1中所述的生化和细胞反应。因此，我们将测试MYPT 1 Thr 696和Thr 696磷酸化对Ca 2+致敏反应都很重要的假设。具体目标3：Ca 2+非依赖性ZIPK是否影响Ca 2+敏化MYPT 1和CPI-17磷酸化？含有ZIPK floxed基因的小鼠将用于成年动物的基因消融，因为该激酶没有选择性药理学抑制剂。我们将测试的假设，Ca 2+非依赖性激酶ZIPK磷酸化MYPT 1和CPI-17作为一个不可分割的组成部分的Ca 2+敏化过程。